Credit: Biodesign Institute

Of all of the paths toward molecular manufacturing, structural DNA nanotechnology seems to provide the most frequent and photogenic advances. By re-engineering the Holliday junction, the basic cross-over structure adapted to build complex structures from DNA, Prof. Hao Yan and his colleagues has been able to construct a variety of new wire frame and mesh structures. A hat tip to ScienceDaily for reprinting this Arizona State University news release “ASU scientists develop innovative twists to DNA nanotechnology“:

In a new discovery that represents a major step in solving a critical design challenge, Arizona State University Professor Hao Yan has led a research team to produce a wide variety of 2-D and 3-D structures that push the boundaries of the burgeoning field of DNA nanotechnology.

The field of DNA nanotechnology utilizes nature’s design rules and the chemical properties of DNA to self-assemble into an increasingly complex menagerie of molecules for biomedical and electronic applications. Some of the Yan lab’s accomplishments include building Trojan horse-like structures to improve drug delivery to cancerous cells, electrically conductive gold nanowires, single molecule sensors and programmable molecular robots.

With their bio-inspired architectural works, the group continues to explore the geometrical and physical limits of building at the molecular level.

“People in this field are very interested in making wire frame or mesh structures,” said Yan. “We needed to come up with new design principles that allow us to build with more complexity in three dimensions.”

In their latest twist to the technology, Yan’s team made new 2-D and 3-D objects that look like wire-frame art of spheres as well as molecular tweezers, scissors, a screw, hand fan, and even a spider web.

The Yan lab, which includes ASU Biodesign Institute colleagues Dongran Han, Suchetan Pal, Shuoxing Jiang, Jeanette Nangreave and assistant professor Yan Liu, published their results in the March 22 issue of Science [abstract].

The twist in their ‘bottom up,’ molecular Lego design strategy focuses on a DNA structure called a Holliday junction.

In nature, this cross-shaped, double-stacked DNA structure is like the 4-way traffic stop of genetics – where 2 separate DNA helices temporality meet to exchange genetic information. The Holliday junction is the crossroads responsible for the diversity of life on Earth, and ensures that children are given a unique shuffling of traits from a mother and father’s DNA.

In nature, the Holliday junction twists the double-stacked strands of DNA at an angle of about 60-degrees, which is perfect for swapping genes but sometimes frustrating for DNA nanotechnology scientists, because it limits the design rules of their structures.

“In principal, you can use the scaffold to connect multiple layers horizontally,” [which many research teams have utilized since the development of DNA origami by Cal Tech's Paul Rothemund in 2006]. However, when you go in the vertical direction, the polarity of DNA prevents you from making multiple layers,” said Yan. “What we needed to do is rotate the angle and force it to connect.”

Making the new structures that Yan envisioned required re-engineering the Holliday junction by flipping and rotating around the junction point about half a clock face, or 150 degrees. Such a feat has not been considered in existing designs.

“The initial idea was the hardest part,” said Yan. “Your mind doesn’t always see the possibilities so you forget about it. We had to break the conceptual barrier that this could happen.”

In the new study, by varying the length of the DNA between each Holliday junction, they could force the geometry at the Holliday junctions into an unconventional rearrangement, making the junctions more flexible to build for the first time in the vertical dimension. Yan calls the backyard barbeque grill-shaped structure a DNA Gridiron.

“We were amazed that it worked!” said Yan. “Once we saw that it actually worked, it was relatively easy to implement new designs. Now it seems easy in hindsight. If your mindset is limited by the conventional rules, it’s really hard to take the next step. Once you take that step, it becomes so obvious.”

The DNA Gridiron designs are programmed into a viral DNA, where a spaghetti-shaped single strand of DNA is spit out and folded together with the help of small ‘staple’ strands of DNA that help mold the final DNA structure. In a test tube, the mixture is heated, then rapidly cooled, and everything self-assembles and molds into the final shape once cooled. Next, using sophisticated AFM and TEM imaging technology, they are able to examine the shapes and sizes of the final products and determine that they had formed correctly.

This approach has allowed them to build multilayered, 3-D structures and curved objects for new applications.

“Most of our research team is now devoted toward finding new applications for this basic toolkit we are making,” said Yan. “There is still a long way to go and a lot of new ideas to explore. We just need to keep talking to biologists, physicists and engineers to understand and meet their needs.”

The video (computer simulation) of the sphere made from DNA, included in the news release, represents an impressive new capability. One thing I like about structural DNA nanotechnology is that every time I think they have just about exhausted the bag of tricks that DNA provides, someone proves me wrong. I look forward to seeing what else they come up with.
—James Lewis, PhD

This 3-D print shows a DNA-based structure designed to test a critical assumption -- that such objects could be realized, as designed, with subnanometer precision. This object is a relatively large, three-dimensional DNA-based structure, asymmetrical to help determine the orientation, and incorporating distinctive design motifs. Subnanometer-resolution imaging with low-temperature electron microscopy enabled researchers to map the object -- which comprises more than 460,000 atoms -- with subnanometer-scale detail. (Credit: Dietz Lab, TU Muenchen)

Before this year the best way to build complex 3D nanostructures from DNA was to use scaffolded DNA origami (see, for example, this post). Last May scientists at the Wyss Institute introduced a DNA tile method for fabricating complex DNA objects that was much faster and much less expensive, and just two weeks ago we posted news that they had extended this method to make arbitrarily complex 3D DNA nanostructures from DNA bricks. Now scientists at the Technische Universität München have published two papers documenting major enhancements to scaffolded DNA origami. From “Reality check for DNA nanotechnology“:

Two major barriers to the advancement of DNA nanotechnology beyond the research lab have been knocked down. This emerging technology employs DNA as a programmable building material for self-assembled, nanometer-scale structures. Many practical applications have been envisioned, and researchers recently demonstrated a synthetic membrane channel made from DNA. Until now, however, design processes were hobbled by a lack of structural feedback. Assembly was slow and often of poor quality. Now researchers led by Prof. Hendrik Dietz of the Technische Universitaet Muenchen (TUM) have removed these obstacles.

One barrier holding the field back was an unproven assumption. Researchers were able to design a wide variety of discrete objects and specify exactly how DNA strands should zip together and fold into the desired shapes. They could show that the resulting nanostructures closely matched the designs. Still lacking, though, was the validation of the assumed subnanometer-scale precise positional control. This has been confirmed for the first time through analysis of a test object designed specifically for the purpose. A technical breakthrough based on advances in fundamental understanding, this demonstration has provided a crucial reality check for DNA nanotechnology.

In a separate set of experiments, the researchers discovered that the time it takes to make a batch of complex DNA-based objects can be cut from a week to a matter of minutes, and that the yield can be nearly 100%. They showed for the first time that at a constant temperature, hundreds of DNA strands can fold cooperatively to form an object — correctly, as designed — within minutes. Surprisingly, they say, the process is similar to protein folding, despite significant chemical and structural differences. “Seeing this combination of rapid folding and high yield,” Dietz says, “we have a stronger sense than ever that DNA nanotechnology could lead to a new kind of manufacturing, with a commercial, even industrial future.” And there are immediate benefits, he adds: “Now we don’t have to wait a week for feedback on an experimental design, and multi-step assembly processes have suddenly become so much more practical.” …

To test the unproven assumption of subnanometer-scale precise positional control, the TUM scientists and their collaborators at MRC Laboratory of Molecular Biology in Cambridge, UK built a large asymmetrical 3D DNA nanostructure incorporating distinctive design motifs, and then characterized its structure with low-temperature electron microscopy. The research was published recently in PNAS (abstract, open access PDF). They designed a DNA nanostructure comprising 15,328 nucleotides (more than 460,000 atoms) assembled from a 7,249-nucleotide long scaffold strand of bacteriophage DNA and 163 short staple strands. The structure formed overnight in a one-pot reaction in high yield. Cryo-electron microscopy enabled a 3D reconstruction based upon tens of thousands of individual images. The resolution of the reconstructed image was sub-nanometer but not quite atomically precise, ranging from 0.97 nm in the core of the nanostructure to 1.4 nm at the periphery. Analysis of the structure determined indicates that the structural order within the nanostructure is comparable to that of natural nanomachines. Detailed comparison of the obtained structure with the designed structure showed more variation than expected in the structure of the DNA helices formed, indicating that the densely packed design led to some unusual DNA topologies. These results indicate that an interactive strategy of designing a folded DNA structure followed by 3D structural analysis will allow construction of a rich variety of precise, complex objects. The authors conclude:

By using chemical groups attached to DNA strands or even reactive motifs formed by DNA itself, this strategy offers an attractive route to achieving complex functionalities known today only from natural nanomachines.

In a second paper just published in Science [abstract], the TUM researchers tackle a major limitation of scaffolded DNA origami: week-long reaction times as the mixture of template and staple DNA strands is very slowly cooled over a very large temperature range, and poor yields. The researchers very carefully followed the rate of structure formation for three different DNA nanostructures as reaction mixes were slowly cooled over a very broad temperature range. One DNA nanostructure was a multilayer platelike structure, one a bricklike object, and a third a gearlike object. They found that the DNA nanostructures each formed at a very narrow temperature range (of about 4° C) that was different for each DNA nanostructure. In addition, the folding was complete in as little as 15 minutes. By choosing the appropriate temperature for each DNA nanostructure, the folding could be complete at constant temperature in as little as 5 minutes. Further, folding at constant temperature greatly increased the yield of correctly folded nanostructures. For several different nanostructures, the increase in yield compared to previous protocols ranged from 7-fold to 330-fold improvement. In absolute terms, the yield of properly folded nanostructres approached 100%. The authors note that several attributes of the folding they observe with their protocols resemble the folding of proteins, despite the chemical and structural differences between proteins and DNA.

From the standpoint of DNA nanotechnology as a component of the Technology Roadmap for Productive Nanosystems, the high yield of well-folded building blocks opens the door to hierarchical assembly of larger objects. It will also greatly facilitate the process of fine-tuning the design of functional molecular machine systems incorporating complex DNA nanostructures. The respective roles to be played by DNA bricks and scaffolded DNA origami, of course, remain to be seen.
—James Lewis, PhD

Computer-generated 3D models (top) and corresponding 2D projection microscopy images (bottom) of nanostructures self-assembled from synthetic DNA strands called DNA bricks. (Image Credit: Yonggang Ke, Wyss Institute, Harvard University.)

This past May we posted news of a major advance in the toolkit for DNA nanotechnology. Researchers led by Wyss Institute core faculty member Peng Yin developed a very versatile, rapid, and inexpensive way to assemble arbitrarily complex 150-nm two-dimensional DNA nanostructures from 42-nucleotide DNA tiles. A hat tip to ScienceDaily for reprinting this Wyss Institute news release of another major advance from the same research group aided by another Wyss Core Faculty member William Shih “Researchers Create Versatile 3D Nanostructures Using DNA ‘Bricks’”:

Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University have created more than 100 three-dimensional (3D) nanostructures using DNA building blocks that function like Lego® bricks — a major advance from the two-dimensional (2D) structures the same team built a few months ago.

In effect, the advance means researchers just went from being able to build a flat wall of Legos®, to building a house. The new method, featured as a cover research article in the 30 November issue of Science [abstract], is the next step toward using DNA nanotechnologies for more sophisticated applications than ever possible before, such as “smart” medical devices that target drugs selectively to disease sites, programmable imaging probes, templates for precisely arranging inorganic materials in the manufacturing of next generation computer circuits, and more. …

Earlier this year, the Wyss team reported in Nature how they could create a collection of 2D shapes by stacking one DNA brick (42 bases in length) upon another.

But there’s a “twist” in the new method required to build in 3D.

The trick is to start with an even smaller DNA brick (32 bases in length), which changes the orientation of every matched-up pair of bricks to a 90 degree angle — giving every two Legos® a 3D shape. In this way, the team can use these bricks to build “out” in addition to “up,” and eventually form 3D structures, such as a 25-nanometer solid cube containing hundreds of bricks. The cube becomes a “master” DNA “molecular canvas”; in this case, the canvas was comprised of 1000 so-called “voxels,” which correspond to eight base-pairs and measure about 2.5 nanometers in size – meaning this is architecture at its tiniest.

The master canvas is where the modularity comes in: by simply selecting subsets of specific DNA bricks from the large cubic structure, the team built 102 3D structures with sophisticated surface features, as well as intricate interior cavities and tunnels. “This is a simple, versatile and robust method,” says Peng Yin, Ph.D., Wyss core faculty member and senior author on the study.

The DNA-brick technique capitalizes on the ability of DNA strands to selectively attach to other strands, thanks to the underlying “recipe” of DNA base pairs. …

Another method used to build 3D structures, called DNA origami, is tougher to use to build complex shapes, Yin said, because it relies on a long “scaffold” strand of DNA that folds to interact with hundreds of shorter “staple” strands – and each new shape requires a new scaffold routing strategy and hence new staples. In contrast, the DNA brick method does not use any scaffold strand and therefore has a modular architecture; each brick can be added or removed independently.

“We are moving at lightning speed in our ability to devise ever more powerful ways to use biocompatible DNA molecules as structural building blocks for nanotechnology, which could have great value for medicine as well as non-medical applications,” says Wyss Institute Founding Director Don Ingber, M.D., Ph.D.

The news release includes a video and an animation showing how the DNA strands self-assemble to build complex 3D objects.

This powerful advance should lead to programmable molecular arrangements for several applications. Any of a great variety of molecular species can be attached to DNA bricks and thus assembled into arbitrarily complex 3D configurations. What sorts of molecular species would give DNA bricks functionality that could be used to build a very primitive nanofactory? Where does this advance stand on the road to molecular manufacturing or productive nanosystems? Back in May of 2005 Chris Phoenix and Tihamer Toth-Fejel authored a report for the NASA Institute for Advanced Concepts (“Large-Product General-Purpose Design and Manufacturing Using Nanoscale Modules“, PDF) in which they proposed two different designs for a very primitive nanofactory based upon planar assembly, each using 5-nm molecular building blocks of unspecified composition, prepared by either chemical synthesis or self-assembly, and incorporating a few simple functional capabilities. Certainly one or more functions could be attached to either these 2.5-nm DNA voxels or the 25-nm larger structures comprising 1000 voxels. Can anyone see a way from this advance to a primitive nanofactory that could be used to build improved nanofactories, leading eventually to molecular manufacturing?
—James Lewis, PhD

(credit: Reck-Peterson Lab, Harvard Medical School)

Among the recommendations of the 2007 Technology Roadmap for Productive Nanosystems is the development of modular molecular composite nanosystems (MMCNs), such as systems in which million-atom-scale DNA frameworks are used to organize various functional molecular components in ways to accomplish specific functions, eventually including atomically precise manufacturing. A step in this direction was taken by Harvard University scientists who used a DNA origami framework as a chasis on which to assemble and test the biological molecular motors that maintain subcellular organization in eukaryotic cells through the organized transport of various molecular cargos. In cells these molecular motors dynein and kinesin transport cargos in opposite directions along a hollow 25-nm-diameter protein track—the microtubule component of the cytoskeleton. In this work, the molecular motors carried a DNA chasis cargo along microtubules for a few tens of micrometers—comparable to the length of a eukaryotic cell. “Tug-of-War in Motor Protein Ensembles Revealed with a Programmable DNA Origami Scaffold” was published online in Science last week [abstract, PDF made available by corresponding author].

The DNA chasis comprised a 12-helix bundle with six DNA double helices on the inside and six on outside of the bundle. A total of 90 unique DNA handles on the outer helices of the chasis, and complementary DNA handles on the cargo-binding domains of the dynein and kinesin motor molecules, made it possible to bind specific motor molecules to specific spots on the DNA chasis. The researchers then measured how fast and how far the DNA chasis cargoes were carried along the microtubules when different ensembles of motor molecules were attached to one DNA chasis. After determining how different number of one motor carried the cargo to one end of the microtubule and different numbers of the other motor carried the cargo to the other end of the microtubule, the researchers tried mixing both motors on one cargo chasis. Not surprisingly, in some cases no movement was observed as the two motors tugged in opposite directions. Specific cleavage of the attachments of one type of motor to the cargo released the cargo to move in the appropriate direction. In summing up their results:

Using DNA origami, we built a versatile, synthetic cargo system that allowed us to determine the motile behavior of microtubule-based motor ensembles. In ensembles of identical-polarity motors, motor number had minimal affect on directional velocity, while ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species. … The system we built pro-vides a powerful platform to investigate the motile properties of any combination of identical- or opposite-polarity motors, and could also be used to investigate the role of motor regulation.

It seems likely that the system reported in this article will enable researchers to learn to regulate the movement of molecular motors carrying very diverse cargos along thick protein tracks to destinations separated by micrometers or ten of micrometers. Would such a capability contribute to the construction of useful molecular assembly lines? Could molecular motors be constructed that move cargos shorter, precisely determined distances along smaller, more precisely arranged tracks? Is this just a cute trick for learning more about biological molecular motors (after all, they do figure prominently in various serious diseases), or could this be a small step toward learning to build nanofactories?
—James Lewis, PhD

Walk traffic man icon assembled by using an atomic force microscope to place molecules of green fluorescent protein (credit: Ludwig-Maximilians University)

Four years ago we cited a report by a German research group of a single molecule cut and paste technology to assemble molecular building blocks on a DNA scaffold. The advance was noteworthy because it combined self-assembly of atomically precise components with the ability to use a manipulator (an atomic force microscope) to place those components at arbitrary positions in a larger structure, analogous to the way in which we use our hands to assemble parts macroscopically. These researchers have extended this technology to arrange single protein molecules. A hat tip to ScienceDaily.com and KurzweilAI.net for pointing to this press release from Ludwig-Maximilians University in Munich “All systems go at the biofactory“:

In order to assemble novel biomolecular machines, individual protein molecules must be installed at their site of operation with nanometer precision. LMU researchers have now found a way to do just that. Green light on protein assembly!

The finely honed tip of the atomic force microscope (AFM) allows one to pick up single biomolecules and deposit them elsewhere with nanometer accuracy. The technique is referred to as Single-Molecule Cut & Paste (SMC&P), and was developed by the research group led by LMU physicist Professor Hermann Gaub. In its initial form, it was only applicable to DNA molecules. However, the molecular machines responsible for many of the biochemical processes in cells consist of proteins, and the controlled assembly of such devices is one of the major goals of nanotechnology. A practical method for doing so would not only provide novel insights into the workings of living cells, but would also furnish a way to develop, construct and utilize designer nanomachines.

In a major step towards this goal, the LMU team has modified the method to allow them to take proteins from a storage site and place them at defined locations within a construction area with nanometer precision. “In liquid medium at room temperature, the “weather conditions” at the nanoscale are comparable to those in a hurricane,” says Mathias Strackharn, first author of the new study. Hence, the molecules being manipulated must be firmly attached to the tip of the AFM and held securely in place in the construction area.

The forces that tether the proteins during transport and assembly must also be weak enough not to cause damage, and must be tightly controlled. To achieve these two goals, the researchers used a combination of antibodies, DNA-binding “zinc-finger” proteins, and DNA anchors. “We demonstrated the method’s feasibility by bringing hundreds of fluorescent GFP molecules together to form a little green man, like the traffic-light figure that signals to pedestrians to cross the road, but only some micrometers high,” Strackharn explains.

With this technique, functional aspects of complex protein machines – such as how combinations of different enzymes interact, and how close together they must be to perform coupled reactions – can be tested directly. A further goal is to develop artificial multimolecular assemblies modeled on natural “cellulosomes”, which could be used to convert plant biomass into biofuels. Strackharn points out the implications: “If we can efficiently build mimics of these ‘enzymatic assembly lines’ by bringing individual proteins together, we could perhaps make a significant contribution to the exploitation of sustainable energy sources.” [abstract of research paper]

The precision achieved in this preliminary report of single-molecule cut-and-paste with proteins is about 10 nm. This is about 100-fold less precise than atomic precision. However, the AFM tip can be moved with atomic precision (0.1 nm). The lower precision seen in this preliminary demonstration is due to the long spacer used to attach the anchor DNA to the surface, so the precision can presumably be improved in future work as it becomes more important.

To use the AFM to build a structure by precisely placing GFP molecules, the Green Fluorescent Protein was fused to a zinc finger protein that binds a specific fragment of double strand DNA. This DNA fragment has a single strand overhang that binds to a single strand anchor DNA on the surface in what the authors refer to as the “unzip” geometry so that the DNA binding can be pulled apart one base pair at a time. The GFP is also fused to a short peptide fragment that will bind to an antibody fragment that is attached to the AFM tip.

The combination of DNA base pair interactions and peptide-antibody interactions allows controlled transfer of the green fluorescent protein-DNA complex from the depot to the AFM tip because only a force of 25 pN is required to separate the complex from the depot one base pair at a time, but a force of 40 pN binds the complex to the AFM tip. Lowering the AFM tip at the target position binds to the anchor DNA at the target, but because the DNA strand at the target position is in the “shear” geometry, this new DNA complex cannot be ruptured by a force of less than 60 pN, so the AFM tip can be retracted leaving the GFP complex in the new position.

Assembling the micrometer-size image of the “traffic man” from individual GFP molecules required 900 steps. Two “traffic man” icons were assembled: one using a red dye coupled to the DNA component of the complex that was assembled in the shape of the sign that means “Don’t walk,” and one using the green fluorescence of the GFP itself in the shape of the sign that means “Walk”. The fact the GFP still fluoresces shows that the forces used during the process are gentle enough to preserve the function of the protein.

This work is an important step in the biology-based folding polymer path toward designing and building complex molecular machine systems (see the Technology Roadmap for Productive Nanosystems for discussions of various paths). With the huge array of molecular machines that biology provides it should be possible to explore the capabilities of some very sophisticated systems. It is not yet clear just how precise this cut and paste system can be made, so it is premature to compare it with other proposed systems of assembling designed arrays of proteins: using DNA origami or DNA tiles as scaffolds.
—James Lewis, PhD

(Credit: Image courtesy of University of California-Los Angeles)

Early this year we commented on progress in designing an artificial enzyme to catalyze the Diels-Alder reaction, an important cycloaddition reaction in synthetic organic chemistry that had been proposed as one strategy to develop molecular building block for molecular manufacturing. A new understanding of exactly how the Diels-Alder reaction occurs validates computational methods that may lead to the ability to design a protein catalyst for whatever reaction is needed. A hat tip to ScienceDaily for reprinting this UCLA news release “New insights into how the most iconic reaction in organic chemistry really works“:

… Now, Kendall N. Houk, UCLA’s Saul Winstein Professor of Organic Chemistry, and colleagues report exactly how the Diels–Alder reaction occurs. Their research is published this week in the early online edition of the journal Proceedings of the National Academy of Sciences [abstract] and will be published in an upcoming print edition.

“We have examined the molecular dynamics of the Diels–Alder reaction, which has become the most important reaction in synthesis, in detail to understand how it happens,” said Houk, who is a member of the California NanoSystems Institute at UCLA.

Houk and his colleagues created a number of simulations — he calls them short movies — of molecules coming together and reacting. …

“The idea,” Houk said, “is to understand how the reaction happens — not just that A goes to B and B goes to C, but to actually follow how the bonds are forming and how the atoms are moving as these things come together. Using the massive computing power we have now, we get a degree of resolution of the mechanism that was not really possible before. It took a lot of computer time, but as a result, we now have unprecedented insight into how this reaction occurs.”

Organic chemists have argued about this for years: If two bonds form during a reaction, do they form at the same time, or does one form first and then the other?

“We find that for the simplest Diels–Alder cycloaddition, it takes only about five femtoseconds on average between the formation of the two bonds; we consider that as occurring simultaneously,” Houk said. (A femtosecond is approximately one millionth of one billionth of a second.)

Houk’s new PNAS paper is his first in the journal since being elected to the National Academy of Sciences in 2010. The same PNAS issue also features an interview with Houk, who is one of the most prolific chemists in the world and one of the world’s leading physical organic chemists.

“We have studied many different classes of reactions and come up with various kinds of rules for understanding why things happen the way they do,” Houk said in the interview.

He and his colleagues — who include David Baker at the University of Washington, Charles Doubleday at Columbia University and Kersey Black at Claremont McKenna College — use computational methods to better understand basic chemical reactions and to design proteins and enzymes to catalyze chemical reactions. The combination of computational design and molecular biology “leads to a catalyst for whatever reaction is needed, if we can get this all to work properly,” Houk said.

Describing his research to predict the structure of novel proteins that could catalyze specific chemical reactions, he said, “The idea is to design a catalyst for any reaction that’s important for whatever reason — an important drug or a commercial product, for example.” …

Or maybe to design catalysts to attach to specific locations on a DNA origami lattice to accomplish a multistep reaction to synthesize some complex molecular building block?
—James Lewis, PhD

Researchers successfully used this nanoparticle, made from several strands of DNA and RNA, to turn off a gene in tumor cells. (credit: : Hyukjin Lee and Ung Hee Lee)

The phenomenon of RNA interference offers one of the most promising therapeutic options of the past decade. Small interfering RNA molecules (siRNAs) specifically decrease the expression of a targeted gene by binding to and destroying the messenger RNA produced by that gene. Delivering these siRNAs to where they are needed is, however, a major challenge. Various types of nanoparticles have shown some success, but a new atomically precise nanoparticle made from DNA and RNA offers better targeting and fewer side effects. A hat tip to KurzweilAI for reprinting this MIT news release “Researchers achieve RNA interference, in a lighter package“:

Using a technique known as “nucleic acid origami,” chemical engineers have built tiny particles made out of DNA and RNA that can deliver snippets of RNA directly to tumors, turning off genes expressed in cancer cells.

To achieve this type of gene shutdown, known as RNA interference, many researchers have tried — with some success — to deliver RNA with particles made from polymers or lipids. However, those materials can pose safety risks and are difficult to target, says Daniel Anderson, an associate professor of health sciences and technology and chemical engineering, and a member of the David H. Koch Institute for Integrative Cancer Research at MIT.

The new particles, developed by researchers at MIT, Alnylam Pharmaceuticals and Harvard Medical School, appear to overcome those challenges, Anderson says. Because the particles are made of DNA and RNA, they are biodegradable and pose no threat to the body. They can also be tagged with molecules of folate (vitamin B9) to target the abundance of folate receptors found on some tumors, including those associated with ovarian cancer — one of the deadliest, hardest-to-treat cancers.

Anderson is senior author of a paper on the particles appearing in the June 3 issue of Nature Nanotechnology [abstract]. Lead author of the paper is former MIT postdoc Hyukjin Lee, now an assistant professor at Ewha Womans University in Seoul, South Korea. …

siRNA-delivering nanoparticles made of lipids, which Anderson’s lab and Alnylam are also developing, have shown some success in turning off cancer genes in animal studies, and clinical trials are now underway in patients with liver cancer. Nanoparticles tend to accumulate in the liver, spleen and lungs, so liver cancer is a natural target — but it has been difficult to target such particles to tumors in other organs.

“When you think of metastatic cancer, you don’t want to just stop in the liver,” Anderson says. “You also want to get to more diverse sites.”

Another obstacle to fulfilling the promise of RNAi has been finding ways to deliver the short strands of RNA without harming healthy tissues in the body. To avoid those possible side effects, Anderson and his colleagues decided to try delivering RNA in a simple package made of DNA. Using nucleic acid origami — which allows researchers to construct 3-D shapes from short segments of DNA — they fused six strands of DNA to create a tetrahedron (a six-edged, four-faced pyramid). A single RNA strand was then affixed to each edge of the tetrahedron.

“What’s particularly exciting about nucleic acid origami is the fact that you can make molecularly identical particles and define the location of every single atom,” Anderson says. …

In their abstract the authors state that “gene silencing occurs only when the ligands are in the appropriate spatial orientation”. Achieving the correct spatial orientation is difficult to do without covalent attachment to an atomically precise nanoparticle.
—James Lewis, PhD

Wyss researchers have built numerals, letters, and a number of other structures using short strands of DNA as building blocks. (Credit: Wyss Institute at Harvard University)

A substantial addition has been made to the toolkit for structural DNA nanotechnology. Currently the only general way to build arbitrarily complex 100-nm-scale DNA objects is scaffolded DNA origami, in which a long (about 7000 bases), biological single stranded DNA molecule is folded into a pre-determined shape through binding to a specially designed set of short, synthetic “staple” strands. A new method now programs self-assembly of arbitrarily complex 150-nm DNA objects from hundreds of distinct single-stranded tiles, each a 42-base strand folded into a 3nm by 7nm tile and attached to four neighboring tiles. With each tile a pixel, the tiles assemble to form a 310-pixel, 150nm-square canvas. A hat tip to ScienceDaily for reprinting this Wyss Institute news release “Wyss Institute Develops New Nanodevice Manufacturing Strategy Using Self-Assembling DNA “Building Blocks”“:

Researchers at the Wyss Institute have developed a method for building complex nanostructures out of short synthetic strands of DNA. Called single-stranded tiles (SSTs), these interlocking DNA “building blocks,” akin to Legos®, can be programmed to assemble themselves into precisely designed shapes, such as letters and emoticons. Further development of the technology could enable the creation of new nanoscale devices, such as those that deliver drugs directly to disease sites.

The technology, which is described in today’s online issue of Nature [abstract], was developed by a research team led by Wyss core faculty member Peng Yin, Ph.D., who is also an Assistant Professor of Systems Biology at Harvard Medical School. Other team members included Wyss Postdoctoral Fellow Bryan Wei, Ph.D., and graduate student Mingjie Dai. …

In focusing on the use of short strands of synthetic DNA and avoiding the long scaffold strand, Yin’s team developed an alternative building method. Each SST is a single, short strand of DNA. One tile will interlock with another tile, if it has a complementary sequence of DNA. If there are no complementary matches, the blocks do not connect. In this way, a collection of tiles can assemble itself into specific, predetermined shapes through a series of interlocking local connections.

In demonstrating the method, the researchers created just over one hundred different designs, including Chinese characters, numbers, and fonts, using hundreds of tiles for a single structure of 100 nanometers (billionths of a meter) in size. The approach is simple, robust, and versatile. …

A short video shows how the single strand tiles assemble to create complex DNA objects.

According to coverage at Physicsworld.com “DNA tiles pave the way“, each shape takes about one hour to produce, compared to a week by the DNA origami technique. In addition it appears to be less expensive since one complete set of tiles costing about £4500 is estimated to be able to make 2 x 10⁹³ possible shapes. With DNA origami a distinct set of staple strands is required for each new shape. On the other hand, yield with the new technique is only 6-40% compared to 95% with DNA origami.

Which technique will prove more convenient for constructing complex systems of molecular machines remains to be seen, but the fact that the new technique is faster and less expensive should speed design-fabricate-test iterations.
—James Lewis, PhD

This illustration shows lithium atoms (red) adhered to a graphene lattice that will produce electricity when bent, squeezed or twisted. Conversely, the graphene will deform when an electric field is applied, opening new possibilities in nanotechnology. Illustration: Mitchell Ong, Stanford School of Engineering

Bulk piezoelectric materials are already used for atomically precise nanopositioning to position the tips of scanning probe microscopes. Would there be any advantages to engineered control of piezoelectrical properties in a two-dimensional material? Currently piezoelectric properties of materials cannot be engineered—it is a property only available in certain 3D crystals. Now calculations have demonstrated that graphene can be made piezoelectric by adsorbing atoms on one surface. A hat tip to Physorg.com for reprinting this Stanford University news release written by Andrew Myers “Straintronics: Engineers create piezoelectric graphene“:

Graphene is a wonder material. It is a one-hundred-times-better conductor of electricity than silicon. It is stronger than diamond. And, at just one atom thick, it is so thin as to be essentially a two-dimensional material. Such promising physics have made graphene the most studied substance of the last decade, particularly in nanotechnology. In 2010, the researchers who first isolated it shared the Nobel Prize.

Yet, while graphene is many things, it is not piezoelectric. Piezoelectricity is the property of some materials to produce electric charge when bent, squeezed or twisted. Perhaps more importantly, piezoelectricity is reversible. When an electric field is applied, piezoelectric materials change shape, yielding a remarkable level of engineering control.

Piezoelectrics have found application in countless devices from watches, radios and ultrasound to the push-button starters on propane grills, but these uses all require relatively large, three-dimensional quantities of piezoelectric materials.

Now, in a paper published in the journal ACS Nano [abstract], two materials engineers at Stanford have described how they have engineered piezoelectrics into graphene, extending for the first time such fine physical control to the nanoscale.

“The physical deformations we can create are directly proportional to the electrical field applied. This represents a fundamentally new way to control electronics at the nanoscale,” said Evan Reed, head of the Materials Computation and Theory Group at Stanford and senior author of the study.

This phenomenon brings new dimension to the concept of ‘straintronics,’ he said, because of the way the electrical field strains—or deforms—the lattice of carbon, causing it to change shape in predictable ways.

“Piezoelectric graphene could provide an unparalleled degree of electrical, optical or mechanical control for applications ranging from touchscreens to nanoscale transistors,” said Mitchell Ong, a post-doctoral scholar in Reed’s lab and first author of the paper.

Using a sophisticated modeling application running on high-performance supercomputers, the engineers simulated the deposition of atoms on one side of a graphene lattice — a process known as doping — and measured the piezoelectric effect.

They modeled graphene doped with lithium, hydrogen, potassium and fluorine, as well as combinations of hydrogen and fluorine and lithium and fluorine on either side of the lattice. Doping just one side of the graphene, or doping both sides with different atoms, is key to the process as it breaks graphene’s perfect physical symmetry, which otherwise cancels the piezoelectric effect.

The results surprised both engineers.

“We thought the piezoelectric effect would be present, but relatively small. Yet, we were able to achieve piezoelectric levels comparable to traditional three-dimensional materials,” said Reed. “It was pretty significant.”

The researchers were further able to fine tune the effect by pattern doping the graphene—selectively placing atoms in specific sections and not others.

“We call it designer piezoelectricity because it allows us to strategically control where, when and how much the graphene is deformed by an applied electrical field with promising implications for engineering,” said Ong.

While the results in creating piezoelectric graphene are encouraging, the researchers believe that their technique might further be used to engineer piezoelectricity in nanotubes and other nanomaterials with applications ranging from electronics, photonics, and energy harvesting to chemical sensing and high-frequency acoustics.

“We’re already looking at new piezoelectric devices based on other 2D and low-dimensional materials, hoping they might open new and dramatic possibilities in nanotechnology,” said Reed.

Could piezoelectric graphene be used with, for example, DNA origami scaffolding to position molecular tools to execute programmed actions? To hear the researchers discussing their work and plans, including possible application to nanomechanical systems, an ACS Nano podcast is available.
—James Lewis, PhD

Image courtesy of the Wyss Institute

“The nanosized robot was created in the form of an open barrel whose two halves are connected by a hinge. The DNA barrel, which acts as a container, is held shut by special DNA latches that can recognize and seek out combinations of cell-surface proteins, including disease markers. This image was created by Campbell Strong, Shawn Douglas, and Gaël McGill using Molecular Maya and cadnano.“

DNA nanotechnology is not only a very promising path toward productive nanosystems and atomically precise manufacturing, but also a path to increasingly sophisticated DNA molecular machines for near-term drug delivery applications in nanomedicine. A recent advance comprises an autonomous DNA nanorobot incorporating a DNA origami chasis and DNA aptamer locks functioning as logical AND gates that are unlocked after the aptamers bind a protein target on the target cell, allowing the nanorobot to discharge its therapeutic cargo. A hat tip to KurzweilAI.net for reprinting this Harvard Gazette news release written by Twig Mowatt “Sending DNA robot to do the job: Technology has potential to seek out cancer cells, cause them to self-destruct“:

Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University have developed a robotic device made from DNA that could potentially seek out specific cell targets within a complex mixture of cell types and deliver important molecular instructions, such as telling cancer cells to self-destruct. Inspired by the mechanics of the body’s own immune system, the technology might one day be used to program immune responses to treat various diseases. The research findings appear today in Science ["A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads" abstract; full text available for fair use on Church lab web site].

Using the DNA origami method, in which complex 3-D shapes and objects are constructed by folding strands of DNA, Shawn Douglas, a Wyss Technology Development Fellow, and Ido Bachelet, a former Wyss postdoctoral fellow who is now an assistant professor in the Faculty of Life Sciences and the Nano-Center at Bar-Ilan University in Israel, created a nanosized robot in the form of an open barrel whose two halves are connected by a hinge. The DNA barrel, which acts as a container, is held shut by special DNA latches that can recognize and seek out combinations of cell-surface proteins, including disease markers. When the latches find their targets, they reconfigure, causing the two halves of the barrel to swing open and expose its contents, or payload. The container can hold various types of payloads, including specific molecules with encoded instructions that can interact with specific cell surface signaling receptors.

Douglas and Bachelet used this system to deliver instructions, which were encoded in antibody fragments, to two different types of cancer cells — leukemia and lymphoma. In each case, the message to the cell was to activate its “suicide switch” — a standard feature that allows aging or abnormal cells to be eliminated. And because leukemia and lymphoma cells speak different languages, the messages were written in different antibody combinations. …

“We can finally integrate sensing and logical computing functions via complex, yet predictable, nanostructures — some of the first hybrids of structural DNA, antibodies, aptamers, and metal atomic clusters — aimed at useful, very specific targeting of human cancers and T-cells,” said George Church, a Wyss core faculty member and professor of genetics at Harvard Medical School, who is principal investigator on the project. …

A key feature of this work is that the DNA aptamer changes structure upon binding its target so it releases its hold on the complementary part of the DNA latch. Since two DNA latches hold the nanorobot in a closed configuration, the latches can be programmed to both respond to the same cell surface target, or to each respond to a different target so that both targets would need to be on the cell to activate the nanorobot to open and allow the payload molecules to bind their targets. This logical AND function allows for much greater specificity in recognizing target cells. As the authors conclude, “These findings demonstrate that the robots can induce a variety of tunable changes in cell behavior.” Conceivably a similar mechanism could be used in an atomically precise manufacturing operation in which DNA nanorobots could add a payload molecule to a workpiece depending on whether both of two specific molecular signals on the workpiece were present.
—James Lewis